1. Field
This disclosure is concerned generally with porous inorganic support materials useful for the immobilization of enzymes. Specifically, this disclosure is concerned with an improved method of regenerating certain porous inorganic support materials which have been used for the immobilization of glucose isomerase.
2. Prior Art
In U.s. Pat. No. 3,868,304, there are disclosed methods of immobilizing glucose isomerase within the pores of highly porous alumina particles to provide a very efficient and reuseable immobilized glucose isomerase system useful for the enzymatic isomerization of glucose (dextrose) to fructose (levulose). As described in that patent, it had been found that the porous alumina carrier for the enzyme should be in particulate form (e.g. within 4-200 mesh) and have an average pore diameter within the range of about 100A to 1000A, preferably within the range of about 180A to about 220A. In patent application Ser. No. 507,209, cited as a related application, an improvement over the alumina carrier is disclosed. The improved glucose isomerase carriers have incorporated thereinto varying amounts of magnesia, the preferred carriers consisting of both alumina and magnesia with the magnesia constituting about 0.84 to 12.0% by weight. Such carriers are referred to herein as MgO-Al.sub.2 O.sub.3 carriers to distinguish them from the Al.sub.2 O.sub.3 carriers.
In using particles of either porous Al.sub.2 O.sub.3 or porous MgO-Al.sub.2 O.sub.3 for the adsorption and, hence, immobilization of glucose isomerase, it has been found that the resulting composites demonstrate a high degree of stability and relatively long enzymatic half-lives. These qualities make the composites commercially attractive since such characteristics are desirable for any large scale conversion of glucose-containing solutions to sweeter fructose-containing solutions. The desirability of being able to continuously and economically convert glucose to fructose is well recognized, especially via enzymatic isomerization methods.
Even though the above-described porous Al.sub.2 O.sub.3 and porous MgO-Al.sub.2 O.sub.3 carriers can be used to prepare immobilized glucose isomerase composites having relatively long half-lives, the use, espeically the continuous use, of such composites is economically time-limited. Regardless of the length of enzymatic half-life of the composites, it can be appreciated that the total enzymatic activity tends to decline with time. Thus, at a given point in time, it becomes uneconomical to continue using the composites because of reduced activity. Accordingly, at that time it becomes more economical to simply replace the spent composite with fresh composite.
Although the above-described porous carriers are relatively inexpensive and may be discarded after use without detracting significantly from the overall favorable economics of using such carriers for glucose isomerase, the reuse of those carriers is highly desirable. Carrier reuse permits yet further economies and also avoids problems associated with discharge of the spent composite. It is known that various pyrolysis treatments can be used to burn off organic constituents on inorganic materials. However, as pointed out in patent application Ser. No. 507,199, cited above, simply pyrolysis does not assure the removal of all contaminants (e.g. various metal ions from the substrate) which tend to minimize subsequent enzyme reloading and half life. In the above patent application, a two step method of regenerating such carrier is disclosed. In the first step, the spent enzyme composite is pyrolyzed at a temperature ranging from about 500.degree. to 900.degree. C under conditions sufficient to remove essentially all carbonaceous material. Then the carrier is reacted with a neutralized citrate solution to assure removal of remaining contaminants. Although the cited two step method is effective in permitting regeneration of the MgO-Al.sub.2 O.sub.3 carriers, it can be appreciated that the pyrolysis step generally requires removal of the spent composite from its container (e.g. a flow through column) and placement in an appropriate furnace. This is followed by removal from the furnace, replacement in the column and treatment with the citrate solution.
We have now found that the above regeneration steps can be replaced with a relatively simple one step regeneration technique which does not require pyrolysis. The regeneration step can be accomplished by fluidizing the spent composite in its original in-use container thereby obviating spent composite removal. Details of our method are described hereunder.